During the previous renewal we made a number of new discoveries and identified several new signaling molecules that interact with Galpha subunits and are expressed in pituitary and other cells. The most significant findings are: 1) Demonstration that Galpha subunits are expressed in excess of Gbetagamma subunits in the Golgi as compared to the plasma membrane (PM); 2) Identification of GAIP, a member of the RGS family that is highly expressed in pituitary cells; 3) Demonstration that GAIP is a component of clathrin coated vesicles (CCVs) and can be phosphorylated by CCVs; 4) Discovery of a novel signaling network composed of GAIP and GIPC (a PDZ domain protein); 5) Discovery of calnuc, a calcium binding protein that is highly expressed in pituitary cells and binds to the C-terminus of Galphai3; 6) Characterization by immunoisolation of a novel smooth ER exit compartment (SEREC). In this renewal application we propose experiments on pituitary cells and other cells designed to follow up several of these findings. Our specific aims are: 1. To determine whether RGS-GAIP functions in endocytosis of G protein coupled receptors (GPCR) and/or in the control of regulated secretion from AtT-20 cells. We will determine whether GPCR and GAIP traffic together after ligand binding, and whether GAIP overexpression affects GPCR trafficking. We will also determine the effect of overexpressing GAIP or antisense GAIP on secretion of ACTH from AtT-20 cells and processing of proteins along the regulated secretory pathway. 2. To investigate the role of GIPC and GAIP in endocytosis of growth factor receptors. Preliminary data suggest that GAIP and GIPC form a complex with the TrkA (NGF) receptor, a receptor tyrosine kinase (RTK). Using a PC-12 cell line stably overexpressing TrkA, we will carry out Co-IP and immunocytochemical experiments to assess whether these molecules colocalize and interact. We will also investigate whether overexpression of GIPC or GAIP affects endocytosis of TrkA and downstream signaling events triggered by binding of ligand to this receptor. 3. To assess the function of calnuc on secretion in AtT-20 pituitary cells. We will overexpress calnuc and calnuc mutants unable to bind Galphai3; and Ca2+ to determine whether increasing or decreasing the levels of expression of calnuc or changing its phosphorylation state affect secretion of ACTH and processing of hormone precursor along the regulated secretory pathway. This work can be expected to provide novel findings and new insights into the mechanisms by which hormone uptake vin via GPCR is controlled and hormone processing and secretion are regulated.